Investigation of the role of novel SGK1 isoforms in regulation of sodium transport in kidney epithelial cells

Abstract

Serum/glucocorticoid regulated kinase 1 (SGK1) is a key component of the pathway that leads to activation of the epithelial sodium channel (ENaC) in the aldosterone-sensitive distal nephron (ASDN). Regulation of ENaC is a major determinant of renal Na+ absorption and overall body fluid homeostasis and blood pressure. Studies from our laboratory have shown that human skin cells express multiple SGK1 isoforms (A-F) that arise from alternative transcriptional start sites and RNA splicing at the SGK1 locus. The aim of this study was to investigate if SGK1 isoforms are also expressed in the ASDN and to assess their potential role in regulating Na+ transport. For these studies the mouse cortical collecting duct cell line mpkCCDcl4, was used as an in vitro model of the ASDN. Comparison of mouse Sgk1 expressed sequence tags (ESTs) with genomic DNA, identified four potential Sgk1 isoforms (Sgk1a-1d). Each isoform has a unique amino terminus of varying size, but otherwise an identical sequence. Using sequence specific primers, mRNA expression of all four isoforms was confirmed by RT-PCR from purified mpkCCDcl4 cell and mouse renal tissue RNA. mpkCCDcl4 cells exposed to aldosterone (Aldo) or Aldo plus insulin (Ins) showed a time-dependent increase in the endogenous expression levels of multiple Sgk1 bands within 1 hour of treatment. These Aldo and Aldo + Ins-induced endogenous Sgk1 bands co-migrated with overexpressed Sgk1 a-d isoform bands. Aldosterone also produced a significant increase in amiloride-sensitive (ENaCmediated) equivalent short circuit current within 2 hours of exposure, peaking after 4 hours. Insulin potentiated the Aldo response, but had no effect alone. Specific inhibitors showed that the hormonal response involved both PI3Kinase and mTOR-dependent and independent pathways. Immunofluorescence studies utilising cloned and tagged SGK1 isoforms in transfected HEK293T cells revealed cytoplasmic network-like staining for all SGK1 isoforms except for SGK1D, which produced plasma membrane staining. In mouse renal tissue, endogenous Sgk1d localised to the basolateral membrane of collecting duct epithelial cells. Furthermore co-immunoprecipitation of cloned human SGK1 and mouse Sgk1 proteins with the Aldo induced regulatory protein, glucocorticoid-induced leucine zipper protein 1 (GILZ1), showed isoform-specific interactions. Collectively, these results build upon our understanding of SGK1 gene expression, protein localisation and function in the ASDN.